Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

De La Cruz, Enrique

Abstract

The Ectonucleotide Pyrophosphatase Phosphodiesterase (ENPP) family members are promiscuous extracellular enzymes that play central roles in human physiology via catalysis of nucleotide and lipid substrates, but remain poorly characterized at the biochemical level. The founding member, ENPP1, is a glycosylated transmembrane enzyme with an extracellular catalytic domain that degrades nucleotide substrates (ATP and cGAMP) into their respective nucleotide monophosphates, playing a key role in both the regulation of biomineralization and innate immune system activation. The ENPP1 ATPase is the major source of extracellular pyrophosphate (PPi), the main chemical inhibitor of calcium phosphate crystal formation in physiology. ENPP1 deficiency lowers systemic PPi levels and induces life-threatening arterial calcifications, which ongoing clinical trials show can be treated by enzyme replacement therapy (ERT) with a soluble ENPP1 biologic. ENPP1 is also the major extracellular cGAMP hydrolase responsible for attenuating activation of the innate immune response through degradation of cGAMP, the stimulator of interferon genes (STING) receptor agonist. Central to understanding the regulatory role of ENPP1 in biomineralization, innate immune system activation, and, more broadly, extracellular purinergic nucleotide signaling is a quantitative understanding of the rate and equilibrium constants for the individual biochemical transitions that define the ATPase and cGAMP hydrolase reaction pathways. Detailed analysis of these reaction mechanisms provides a rigorous framework for understanding ENPP enzyme function and helps develop testable hypotheses regarding physiological function and molecular mechanisms of other ENPP enzymes. The work presented here provides a kinetic analysis of the ENPP1 catalytic ATPase cycle, revealing intrinsic regulatory mechanisms of ENPP1 crucial to function within its physiological environment, as well as the beginnings of a kinetic analysis of the ENPP1 cGAMP hydrolase activity.

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